A dual-therapy approach for the treatment of biofilm-mediated Salmonella gallbladder carriage.

Asymptomatic carriage of Salmonella Typhi continues to facilitate the transmission of typhoid fever, resulting in 14 million new infections and 136,000 fatalities each year. Asymptomatic chronic carriage of S. Typhi is facilitated by the formation of biofilms on gallstones that protect the bacteria from environmental insults and immune system clearance. Here, we identified two unique small molecules capable of both inhibiting Salmonella biofilm growth and disrupting pre-formed biofilm structures without affecting bacterial viability. In a mouse model of chronic gallbladder Salmonella carriage, treatment with either compound reduced bacterial burden in the gallbladder by 1-2 logs resulting in bacterial dissemination to peripheral organs that was associated with increased mortality. Co-administration of either compound with ciprofloxacin not only enhanced compound efficacy in the gallbladder by a further 1-1.5 logs for a total of 3-4.5 log reduction, but also prevented bacterial dissemination to peripheral organs. These data suggest a dual-therapy approach targeting both biofilm and planktonic populations can be further developed as a safe and efficient treatment of biofilm-mediated chronic S. Typhi infections.

Cyclic electron flow (CEF) and ascorbate pathway activity provide constitutive photoprotection for the photopsychrophile, Chlamydomonas sp. UWO 241 (renamed Chlamydomonas priscuii).

Under environmental stress, plants and algae employ a variety of strategies to protect the photosynthetic apparatus and maintain photostasis. To date, most studies on stress acclimation have focused on model organisms which possess limited to no tolerance to stressful extremes. We studied the ability of the Antarctic alga Chlamydomonas sp. UWO 241 (UWO 241) to acclimate to low temperature, high salinity or high light. UWO 241 maintained robust growth and photosynthetic activity at levels of temperature (2 °C) and salinity (700 mM NaCl) which were nonpermissive for a mesophilic sister species, Chlamydomonas raudensis SAG 49.72 (SAG 49.72). Acclimation in the mesophile involved classic mechanisms, including downregulation of light harvesting and shifts in excitation energy between photosystem I and II. In contrast, UWO 241 exhibited high rates of PSI-driven cyclic electron flow (CEF) and a larger capacity for nonphotochemical quenching (NPQ). Furthermore, UWO 241 exhibited constitutively high activity of two key ascorbate cycle enzymes, ascorbate peroxidase and glutathione reductase and maintained a large ascorbate pool. These results matched the ability of the psychrophile to maintain low ROS under short-term photoinhibition conditions. We conclude that tight control over photostasis and ROS levels are essential for photosynthetic life to flourish in a native habitat of permanent photooxidative stress. We propose to rename this organism Chlamydomonas priscuii.

The Abundance and Organization of Salmonella Extracellular Polymeric Substances in Gallbladder-Mimicking Environments and In Vivo.

Salmonella enterica serovar Typhi causes chronic infections by establishing biofilms on cholesterol gallstones. The production of extracellular polymeric substances (EPSs) is key to biofilm development, and biofilm architecture depends on which EPSs are made. The presence and spatial distribution of Salmonella EPSs produced in vitro and in vivo were investigated in Salmonella enterica serovar Typhimurium and S. Typhi biofilms by confocal microscopy. Comparisons between serovars and EPS-mutant bacteria were carried out by examining growth on cholesterol-coated surfaces, with human gallstones in ox or human bile, and in mice with gallstones. On cholesterol-coated surfaces, no major differences in EPS biomass were found between serovars. Cocultured biofilms containing wild-type (WT) and EPS-mutant bacteria demonstrated WT compensation for EPS mutations. Analysis of biofilm EPSs from gallbladder-mimicking conditions found that culture in human bile more consistently replicated the relative abundance and spatial organization of each EPS on gallstones from the chronic mouse model than culture in ox bile. S. Typhimurium biofilms cultured in vitro on gallstones in ox bile exhibited colocalized pairings of curli fimbriae/lipopolysaccharide and O-antigen capsule/cellulose, while these associations were not present in S. Typhi biofilms or in mouse gallstone biofilms. In general, the inclusion of human bile with gallstones in vitro replicated biofilm development on gallstones in vivo, demonstrating the strength of this model for studying biofilm parameters or EPS-directed therapeutic treatments.

Ceftobiprole Medocaril Is an Effective Post-Exposure Treatment in the Fischer 344 Rat Model of Pneumonic Tularemia.

Francisella tularensis subspecies tularensis is a category-A biothreat agent that can cause lethal tularemia. Ceftobiprole medocaril is being explored as a medical countermeasure for the treatment of pneumonic tularemia. The efficacy of ceftobiprole medocaril against inhalational tularemia was evaluated in the Fischer 344 rat model of infection. The dose was expected to be effective against F. tularensis isolates with ceftobiprole minimum inhibitory concentrations ≤0.5 µg/mL. Animals treated with ceftobiprole medocaril exhibited a 92% survival rate 31 days post-challenge, identical to the survival of levofloxacin-treated rats. By comparison, rats receiving placebo experienced 100% mortality. Terminally collected blood, liver, lung, and spleen samples confirmed disseminated F. tularensis infections in most animals that died prior to completing treatments (placebo animals and a rat treated with ceftobiprole medocaril), although levels of bacteria detected in the placebo samples were significantly elevated compared to the ceftobiprole-medocaril-treated group geometric mean. Furthermore, no evidence of infection was detected in any rat that completed ceftobiprole medocaril or levofloxacin treatment and survived to the end of the post-treatment observation period. Overall, survival rates, body weights, and bacterial burdens consistently demonstrated that treatment with ceftobiprole medocaril is efficacious against otherwise fatal cases of pneumonic tularemia in the rat model.

Salmonella Biofilms Tolerate Hydrogen Peroxide by a Combination of Extracellular Polymeric Substance Barrier Function and Catalase Enzymes.

The ability of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) to cause chronic gallbladder infections is dependent on biofilm growth on cholesterol gallstones. Non-typhoidal Salmonella (e.g. S. Typhimurium) also utilize the biofilm state to persist in the host and the environment. How the pathogen maintains recalcitrance to the host response, and oxidative stress in particular, during chronic infection is poorly understood. Previous experiments demonstrated that S. Typhi and S. Typhimurium biofilms are tolerant to hydrogen peroxide (H2O2), but that mutations in the biofilm extracellular polymeric substances (EPSs) O antigen capsule, colanic acid, or Vi antigen reduce tolerance. Here, biofilm-mediated tolerance to oxidative stress was investigated using a combination of EPS and catalase mutants, as catalases are important detoxifiers of H2O2. Using co-cultured biofilms of wild-type (WT) bacteria with EPS mutants, it was demonstrated that colanic acid in S. Typhimurium and Vi antigen in S. Typhi have a community function and protect all biofilm-resident bacteria rather than to only protect the individual cells producing the EPSs. However, the H2O2 tolerance deficiency of a O antigen capsule mutant was unable to be compensated for by co-culture with WT bacteria. For curli fimbriae, both WT and mutant strains are tolerant to H2O2 though unexpectedly, co-cultured WT/mutant biofilms challenged with H2O2 resulted in sensitization of both strains, suggesting a more nuanced oxidative resistance alteration in these co-cultures. Three catalase mutant (katE, katG and a putative catalase) biofilms were also examined, demonstrating significant reductions in biofilm H2O2 tolerance for the katE and katG mutants. Biofilm co-culture experiments demonstrated that catalases exhibit a community function. We further hypothesized that biofilms are tolerant to H2O2 because the physical barrier formed by EPSs slows penetration of H2O2 into the biofilm to a rate that can be mitigated by intra-biofilm catalases. Compared to WT, EPS-deficient biofilms have a heighted response even to low-dose (2.5 mM) H2O2 challenge, confirming that resident bacteria of EPS-deficient biofilms are under greater stress and have limited protection from H2O2. Thus, these data provide an explanation for how Salmonella achieves tolerance to H2O2 by a combination of an EPS-mediated barrier and enzymatic detoxification.

Salmonella Extracellular Polymeric Substances Modulate Innate Phagocyte Activity and Enhance Tolerance of Biofilm-Associated Bacteria to Oxidative Stress.

Salmonella enterica serovar Typhi causes 14.3 million acute cases of typhoid fever that are responsible for 136,000 deaths each year. Chronic infections occur in 3%-5% of those infected and S. Typhi persists primarily in the gallbladder by forming biofilms on cholesterol gallstones, but how these bacterial communities evade host immunity is not known. Salmonella biofilms produce several extracellular polymeric substances (EPSs) during chronic infection, which are hypothesized to prevent pathogen clearance either by protecting biofilm-associated bacteria from direct humoral attack or by modulating innate phagocyte interaction with biofilms. Using wild-type and EPS-deficient planktonic and biofilm Salmonella, the direct attack hypothesis was tested by challenging biofilms with human serum and antimicrobial peptides. Biofilms were found to be tolerant to these molecules, but these phenotypes were independent of the tested EPSs. By examining macrophage and neutrophil responses, new roles for biofilm-associated capsular polysaccharides and slime polysaccharides were identified. The S. Typhi Vi antigen was found to modulate innate immunity by reducing macrophage nitric oxide production and neutrophil reactive oxygen species (ROS) production. The slime polysaccharides colanic acid and cellulose were found to be immune-stimulating and represent a key difference between non-typhoidal serovars and typhoidal serovars, which do not express colanic acid. Furthermore, biofilm tolerance to the exogenously-supplied ROS intermediates hydrogen peroxide (H2O2) and hypochlorite (ClO) indicated an additional role of the capsular polysaccharides for both serovars in recalcitrance to H2O2 but not ClO, providing new understanding of the stalemate that arises during chronic infections and offering new directions for mechanistic and clinical studies.

Salmonella Biofilm Formation, Chronic Infection, and Immunity Within the Intestine and Hepatobiliary Tract.

Within the species of Salmonella enterica, there is significant diversity represented among the numerous subspecies and serovars. Collectively, these account for microbes with variable host ranges, from common plant and animal colonizers to extremely pathogenic and human-specific serovars. Despite these differences, many Salmonella species find commonality in the ability to form biofilms and the ability to cause acute, latent, or chronic disease. The exact outcome of infection depends on many factors such as the growth state of Salmonella, the environmental conditions encountered at the time of infection, as well as the infected host and immune response elicited. Here, we review the numerous biofilm lifestyles of Salmonella (on biotic and abiotic surfaces) and how the production of extracellular polymeric substances not only enhances long-term persistence outside the host but also is an essential function in chronic human infections. Furthermore, careful consideration is made for the events during initial infection that allow for gut transcytosis which, in conjunction with host immune functions, often determine the progression of disease. Both typhoidal and non-typhoidal salmonellae can cause chronic and/or secondary infections, thus the adaptive immune responses to both types of bacteria are discussed with particular attention to the differences between Salmonella Typhi, Salmonella Typhimurium, and invasive non-typhoidal Salmonella that can result in differential immune responses. Finally, while strides have been made in our understanding of immunity to Salmonella in the lymphoid organs, fewer definitive studies exist for intestinal and hepatobiliary immunity. By examining our current knowledge and what remains to be determined, we provide insight into new directions in the field of Salmonella immunity, particularly as it relates to chronic infection.

Chronic biofilm-based infections: skewing of the immune response.

Many of the deadliest bacterial diseases that plague humanity in the modern age are caused by bacterial biofilms that produce chronic infections. However, most of our knowledge of the host immune response comes from the study of planktonic pathogens. While there are similarities in the host response to planktonic and biofilm bacteria, specific immune responses toward biofilms have not been well studied; the only apparent difference is the inability to clear the bacteria allowing the biofilm infection to become chronic. In some cases, the biofilms skew T-cell response toward a balance that allows a stalemate between the host and the pathogen, in which the infection can become persistent. In this minireview, we will summarize well-known examples of this phenomena as well as some emerging studies that may indicate that this situation is much more common than initially thought.